The compound ECO-04601 (hereinafter ECO-4601) is a novel farnesylated dibenzodiazepinone, namely 10-farnesyl-4,6,8-trihydroxy-dibenzodiazepin-11-one, having the structure of Formula I as shown below.

ECO-4601 has been isolated from fermentation cultures of a novel strain of the actinomycete Micromonospora sp. as disclosed in U.S. patent application Ser. No. 10/762,107 filed Jan. 21, 2004 (published Feb. 24, 2005 as US 2005-0043297 A1, issued as U.S. Pat. No. 7,101,872 on Sep. 5, 2006), also published Aug. 5, 2004 as PCT International Application WO2004/065591. The structure of ECO-4601 has been subsequently disclosed by Charan et al. (2004), J. Nat. Prod., vol. 67, pages 1431-1433, the compound having been extracted from solids collected from a fermentation broth of Micromonospora strain DPJ12 that was isolated from the marine ascidian Didemnum proliferum Kott. at Shishijima Island, Japan. Charan et al. have referred to the isolated farnesylated dibenzodiazepinone as “diazepinomicin”. The compound ECO-4601 has also been subsequently disclosed by Igarashi et al. (2005), J. Antibiot., vol. 58, pages 350-352, this group having isolated the compound by extraction of a fermentation broth of a culture of an actinomycete, Micromonospora sp. TP-A086, that was isolated from a soil sample collected in Osawano, Japan. ECO-4601 has been shown to possess anti-bacterial and anti-cancer activities, and results from animal model studies indicate an in vivo anti-cancer potency of ECO-4601 (see U.S. patent application Ser. No. 10/951,436 filed Sep. 27, 2004 (published May 19, 2005 as US 2005-010736 A1, issued as U.S. Pat. No. 7,186,713 on Mar. 6, 2007), and U.S. patent application Ser. No. 11/130,295 filed May 16, 2005 (published Apr. 13, 2006 as US 2006-0079508 A1)).
Production of limited amounts of ECO-4601 via fermentation methods have been disclosed. U.S. patent application Ser. No. 11/130,295 discloses that ECO-4601 can be produced by inoculating a culture medium with a sample of a microorganism capable of producing ECO-4601, such as Micromonospora strain 046-ECO11 or strain [S01]046, and thereafter incubating the inoculated culture with aeration by agitation such as by shaking on a water bath or in a fermentor, or by injection of air, oxygen or an appropriate gaseous mixture into the culture medium. Incubation can last for a period of about 3 to 4 days at temperatures of about 18 to about 40° C., and at a pH of about 6 to about 9, in a suitable medium containing assimable sources of carbon, nitrogen, optional inorganic salts and other known growth factors. Upon completing cultivation, ECO-4601 can be isolated from the culture medium by techniques known in the art such as centrifugation, adsorption, chromatography, and filtration. Organic solvents such as ethyl acetate, n-butanol, n-butyl acetate or 4-methyl-2-pentanone may be mixed with the cultivated culture medium and the organic layer separated by centrifugation followed by solvent removal by evaporation to dryness, or by evaporation to dryness under a vacuum, to yield an ECO-4601 containing residue. Downstream purification can be performed by optionally reconstituting the residue with ethanol, ethyl acetate, methanol or a mixture thereof, and re-extraction in a two-phase system using a suitable organic solvent such as hexane, acetonitrile, ethyl acetate, methanol and carbon tetrachloride, methylene chloride or a mixture thereof, followed by further purification by techniques known in the art, such as chromatography.
Fermentation methods for producing limited amounts of the farnesylated dibenzodiazepinone of the present invention have been described in the art. For example, Igarashi et al. (supra) described a process wherein a 5 liter fermentation broth was extracted with 1-butanol and the organic layer concentrated in vacuo to yield an oily extract of 17 grams. Chromatographic purification (silica gel followed by LH-20) of the extract yielded 16 mg of light yellow needles. In Charan et al. (supra), a process was described wherein a 4 liter fermentation broth of Micromonospora DPJ12 was centrifuged and the resulting cell mass and HP20 resin (50 g/L of broth) were washed with water and extracted with methanol (3×250 mL). The combined methanol extracts were concentrated in vacuo and extracted with ethyl acetate (4×60 mL) to yield an extract of 278 milligrams. Further purification (Sephadex LH-20 column chromatography followed by reversed-phase HPLC) yielded 4.8 mg of the diazepinomicin product.
For development of ECO-4601 into a commercial pharmaceutical product, fermentation production of the compound will be required on a scale(s) to generate quantities of the compound to meet sufficiently supply requirements for clinical trial testing regimens, and thereafter for subsequent commercial manufacturing of formulations and dosages that will be prescribed to patients in need of treatment. As such, there remains the need for a robust and commercially-practical fermentation process pertaining to the fermentation of Micromonospora sp. for the production of quantities of ECO-4601 useful for clinical trials and commercial production of pharmaceutical products. Advantageously, the fermentation process should provide several benefits in terms of being scalable from a laboratory level to various levels commercial production, while concomitantly not requiring an increased number of fermentation vessels or other pieces of equipment and/or steps involved in downstream processing and purification steps, nor require increased volumes of an extraction solvent or solvents beyond that directly proportional to an increased fermentation volume. As well, there exists a need for a fermentation process for the production of ECO-4601 that provides for a high yield rate of the compound from a fermentation broth, and which allows for the compound to be efficiently extracted from the fermentation broth and provided in a form, preferably a concentrated form, that is stable, easily transportable, and which can thereafter be easily subjected to downstream processing for production of crystalline forms of ECO-4601 that may thereafter be formulated into pharmaceutically-acceptable compositions for use in patients requiring need of such compositions.